Are genes within creatures such as bacteria
capable of evolving unique and novel functions via mutations and natural
selection?The short answer is - Yes.However, such
evolution, although capable of yielding many startling new abilities, is limited
to the lowest levels of functional complexity (like antibiotic resistance).

The de novo evolution of antibiotic resistance
is based on the specificity of antibiotic interactions with various
protein sequences within a bacterium. Because of the specificity of such
interactions, a very high ratio of mutations are able to interfere with or
completely disrupt these specific interactions - and antibiotic resistance is
the result. In other words, there is a relatively high likelihood
that a particular mutation in a target sequence will result in antibiotic
resistance. This high likelihood translates into a very rapid evolutionary
process. In real life, this is exactly what happens. The evolution
of antibiotic resistance of any previously susceptible bacterial colony to just
about any antibiotic is usually realized within a very short period of sustained
antibiotic exposure.

This paper begins with the historical discovery
of antibiotics and the rapid arrival of resistant strains of bacteria to these
antibiotics.Also, the specific mechanisms of antibiotic action as well as resistance
mechanisms are discussed - to include limits beyond which the powers of
evolution seem to fail.

The
antibiotic age was ushered in with the accidental discovery of penicillin by
Alexander Fleming (1881-1955) in 1928. Even though it was over ten years before
mass production of penicillin was achieved, a new era had arrived. Antibiotics
proved to be wonder drugs in that they killed infection by bacteria without
significantly harming the host, if at all. This was a first in medicine. Never
before had nature and sickness seemed so much within the control mankind.

The euphoria was short lived however.
Shortly after the general usage of antibiotics began the microscopic world
revealed its genius for becoming resistant to antibiotics. It truly seemed like
a fulfillment of Darwin's prophetic vision and the firm establishment of his
Theory of Evolution as unshakable. Today, Darwin's Theory of Evolution seems
more validated than ever by these little creatures. The early advances made by
antibiotic and antiviral medications seem to be almost completely overcome by
the continued evolution of antibiotic and antiviral resistance. So called
"Superbugs" are springing up everywhere that are resistant to every antibiotic
or antiviral currently known to man. Evolution seems not only to be a wonderful,
but also a terrible reality. But is it all really as it seems?

Bacteria become resistant to
antibiotics by a very simple method of natural selection. When a large number of
bacteria are presented for the first time with an antibiotic, most, if not all
of them, die off. If all of them die, then obviously no resistance is
gained for that particular bacterial colony or group. The problem is that
sometimes one or two or even a few bacteria survive the initial exposure. This
is because they were previously resistant before exposure to the antibiotic. Of
course, after they survive the initial exposure they reproduce themselves and
make a new colony of bacteria. Now, every bacterium in that colony is a clone of
the original resistant bacterium and so all of them are resistant to that
particular antibiotic to the same degree. But how did the first one or two
resistant bacteria survive to pass on their evolved resistance to their
offspring?

There are three main targets that
antibiotics attack:

Bacterial protein synthesis

Bacterial nucleic acid replication and repair

Cell wall biosynthesis enzymes and substrates

So, intuitively there are also three basic mechanisms of
bacterial antibiotic resistance:

Alteration of the antibiotic target

Restriction of antibiotic access to the target

Direct inactivation of the antibiotic

Obviously then, if a bacterium can achieve any
of these three blocks to the activity of an antibiotic, it is resistant to that
antibiotic. If this ability is due to a genetic alteration, then this
alteration will be passed on to each and everyone of its offspring since
bacteria reproduce in a clonal fashion. In short, this is evolution in action.

Obviously, bacterial antibiotic resistance is
achieved in many ways to include spontaneous genetic mutations that result in
new traits or functions that were not present in previous generations of that
bacterial group or species.New beneficial functions did in fact "evolve" spontaneously through
mutation and natural selection in such cases.Clearly then, the process of evolution is actually demonstrated by these
little creatures - or is it?Some argue that these
changes are not "evolutionary" since they do not really increase the information
content of the DNA within that bacterium. Consider the following comments by Dr.
Carl Wieland (An Australian Medical Doctor):

This misconception [about
antibiotic resistance and evolution] may be partly due to the fact that even
many science graduates believe that the mechanism of antibiotic resistance
involves the acquisition of new DNA information by accidental mutations... But
resistance does not normally arise like this.
Loss of control over an enzyme's production
can engender antibiotic resistance. Take for instance penicillin resistance in
Staphylococcus bacteria. This requires the bacterium to have DNA
information coding for production of a complicated enzyme (penicillinase), which
specifically destroys penicillin. It is extremely unlikely that such complex
information could arise in a single mutation step, and in fact it does not.
Mutation can cause the loss of control of its production, so much greater
amounts are produced, and a bacterium producing large quantities of
penicillinase will survive when placed in a solution containing penicillin,
whereas those producing lesser amounts will not. The information for producing
this complicated chemical was, however, already present 1

Is Dr. Wieland correct in his statement here?Actually, as described in more detail below, Wieland is at least partly
incorrect.Point mutations to the penicillin binding
proteins (PBPs) can and do result in penicillin resistance even in the absence
of the penicillinase gene.It may be true that penicillinase production is the most significant
mechanism of penicillin resistance in gram-positive bacteria, but clearly this
is not the only mechanism.The â€œevolutionâ€ of
new resistance mechanisms via the mutation of existing genes does in fact occur.
However, Wieland is correct in saying that some mutations can result in an
increased production of a pre-existing penicillinase enzyme from a pre-existing
penicillinase gene.He is also correct in stating
that the information for producing the penicillinase enzyme does not
spontaneously evolve in those bacteria that are able to produce it, but was
already present either through vertical transmission from the previous
generation or via horizontal transmission through the action of plasmids or
other methods of DNA transfer between bacteria.

Also, although no one has ever observed the de
novo evolution of a penicillinase enzyme, evolutionary scientists present
evidence for the original evolution of penicillinase from existing bacterial
genes - but this still remains hypothetical until such proposed evolutionary
pathways can be demonstrated in real time. So far, not even
a single hypothesized step in the pathway of penicillinase evolution has ever
been demonstrated to actually evolve in any bacterium (see appendix).

Certainly
there are many similarities, especially
when one considers the three-dimensional structures and active sites between
homologous sequences that are supposed to be the most likely precursor proteins
to penicillinase (appendix), but there might be a few functionally significant
neutral gaps between them. How are these neutral gaps overcome?
Ultimately, if the crossing of such evolutionary paths between these two enzymes
is truly an easy process, as many claim it is, then why is beta-lactamase
evolution not being demonstrated in the lab? If it is so easy, then why is
it so difficult to demonstrate?

Similar evolutionary limits have been found with
other single protein enzymes - like lactase
evolution experiments showing "limited evolutionary potential" in mutant forms
of K12 E. coli bacteria. It seems like the average distance of
selectable lactase sequences in sequence space is quite significant given the
lack of lactase evolution over the course of many tens of thousands of
generations in a lactose rich environment. The same limitations seem to be
present when it comes other functions that exist at a similar level of
functional complexity (i.e., having a similar minimum sequence size and
specificity of amino acid residue arrangement for a minimal degree of selectable
function).

Of course, some beta-lactamases, such as the
class C beta-lactams, maintain some DD-peptidase activity.
This might be a very good clue to the existence of a real evolutionary pathway,
but not necessarily. Neutral gaps can still exist even if the other
functions from proposed enzymes of origin exist in the "target" enzyme.
The problem for the class C beta-lactams is that their DD-peptidase function is
not great enough to produce a selective advantage as far as DD-peptidase
function is concerned. Several specific mutations are required before a
level of selectively advantageous DD-peptidase function can be realized.
One or more of these specific mutations are most certainly neutral as far as
their functional selective advantage is concerned.
Also, the reverse is not necessarily true. Functional DD-peptidases do not
have beta-lactamase activity. In the proposed evolutionary pathway from a
DD-peptidase to a beta-lactamase, each and every step needs to increase
beta-lactamase activity in a selectable way or there will be a neutral or even
detrimental block in the pathway.

A neutral change is a change in the genetic
sequence (genotype) that cannot be distinguished by natural selection from
different genetic sequences that have that same function (phenotype).
The problem is that with every additional neutral mutation that is
required along a path toward new function, the average time required to traverse
this path increases exponentially. This neutral gap problem seems
to be the most likely source of "limited evolutionary potential" when it comes
to evolving novel functions - like single protein enzymes (i.e., penicillinase).

So yes, it is statistically possible but
improbable that penicillinase evolution is responsible for anything as far as "de
novo" penicillin resistance within a newly resistant population.
Penicillinase, when detected in a bacterial population, was most likely already
there before the selection pressures of penicillin antibiotics were applied to
that population.In other words, penicillinase most
likely existed in the genomes of bacteria long before Alexander Fleming came on
the scene.

The same can probably be said of many plant,
insect, rodent, and other "weed and pest" resistance to the chemicals used to
kill them off.Consider the following quote from the geneticist Francisco Ayala:

The genetic variants required for
resistance to the most diverse kinds of pesticides were apparently present in
every one of the populations exposed to these man-made compounds.3

As additional support for this statement,
consider that bacteria recovered from historical isolation have been found to be
resistant to modern antibiotics. In 1988 bacteria were recovered from the colons
(intestines) of Arctic explorers who froze where they died in 1845. Many decades
later, these explorers where found and various studies where done on their
bodies. Bacteria from their intestines were actually grown and subjected to
various modern antibiotic medications. Many of the bacterial colonies grown were
found to be resistant to many modern antibiotics, proving that this resistance
did not evolve over just the past 60 years or so since the antibiotic age began,
but where already present before humans started using antibiotics to fight
bacterial infections. 4

These methods of resistance
are not limited to bacteria, but extend to the plant and animal kingdoms as
well. Rats, for example, have been shone to be able to develop a resistance to
the poison warfarin. In the 1950's, England was using warfarin to kill off its
rat population. In a few years, the rats had become resistant to warfarin. But
how did they do it? Warfarin kills rats by inhibiting an enzyme involved
in the metabolism of vitamin K. Since Vitamin K is vital for life, rats who lost
the ability to synthesize vitamin K in their cells died. Warfarin-resistant rats
were found to have a mutated form of the enzyme that warfarin inhibited. The
different shape and structure of the enzyme prevented warfarin from binding and
blocking Vitamin K synthesis. These mutant rats were still able to
synthesize enough vitamin K to live.2

Does this sound familiar?
Many times bacteria achieve antibiotic resistance via the same kinds of
mutations - though sometimes the mutated enzymes are not as efficient as the
non-mutated or "wild-type" forms. For example,
the mutated enzyme in rats that blocks the activity of warfarin is 10 times less
efficient at synthesizing a given amount of vitamin K.
Obviously then, if the warfarin was removed from the environment, those rats
with this less efficient enzyme would not be as "fit" as those with the original
wild-type form of this enzyme. Without warfarin in the environment, these
rats would be quickly replaced by rats with the original enzyme unless the
mutant rats already evolved "compensatory mutations" - which can, over time,
overcome the lessened effects of the mutant enzyme so that it can also achieve
"wild-type" levels of activity in many cases.48

The same thing happens with
bacterial colonies when a particular antibiotic is removed from their
environment. Sometimes they revert back to the original wild-type sequence
and again become "sensitive" to the original antibiotic. In fact, due to
this phenomenon, many hospitals are considering the use of cyclic antibiotic
formularies where antibiotics would be rotated after a period of time. One
type of antibiotic would be used for a while, and then replaced by a different
type of antibiotic just as the bacterial population was gaining resistance.
After a while, the second antibiotic would be replaced by a third type of
antibiotic - and so on. Then after a bit more time, the original
antibiotic could be used again will full effect and the cycle would start over.

There is just one little problem
with this idea. The problem is that many of the disadvantages sustained by
the initial evolution against many types of antibiotics can be compensated for
by "compensatory mutations".48 In other words, bacteria that
take on certain disadvantages in order to gain a particularly vital mutational
advantage can often compensate for the disadvantages at a later time with
additional mutations that re-enhance the function that was suppressed by the
initial detrimental aspects of the original mutation(s).

Still, such novel functional
changes are only found at the lowest levels of functional complexity where no
more than a few hundred amino acid residue "parts" or "characters" are required
to work together in a fairly specified arrangement. Any novel function
requiring more than a few hundred fairly specified amino acids working together
at the same time has never been observed to evolve in real time. Obviously
there must be some blockade that slows evolutionary progress down in an
exponential manner with increasing functional complexity.

Herbicides also work the same way.
Consider the following quote from geneticist Maceirj Giertych (Ph.D., D.Sc.):

Much evolutionary publicity is
attached to forms that develop resistance to man-made chemicals. Usually they
are variants that exist in nature but were selected out by the chemical reagent.In one instance, it was demonstrated that a single
nucleotide substitution in the genome was responsible for resistance to a
weed-specific herbicide. The herbicide is 'custom-made' for attachment and
deactivation of a vital protein specific for the weed plant. A single change in
the genetic code for this protein, in the sector used for defining the herbicide
attachment, deprives the herbicide of attachability and therefore of its
herbicidal properties. Such a change has no selective value except in the
context of the man-made herbicide. 5

Viruses also evolve using similar techniques.
Various mutations in the viral genome result in blocks to antiviral binding
sites - just like the blocks to antibiotic binding sights.These blocks may result in a change to the viral protein coat.Such changes may prevent antibody recognition.It all depends upon the specific action and target of the antiviral, but
the principle is the same.A minimal change to the
target gives significant resistance to the host. This phenomenon is made possible because of the high level of
specificity between the antibody / antibiotic / antiviral etc. and their target
sequences.51

But, what about the argument that evolution only
takes away information or that all mutations are harmful in one way or another?Some argue that resistant bacteria are less "fit" than their "wild-type"
counterparts and that their pathogenicity as well as their metabolic and
reproduction rates are lower than before?Consider the following quote from Wieland:

So-called 'supergerms' in
hospitals are not 'super' at all. What has happened is that the use of
antibiotics in modern hospitals has meant that the only ones surviving
are
those, which have all the resistance factors. If a person gets a serious
infection with one of these resistant types, the infection is not therefore more
aggressive than if it was a non-resistance form of the same bug; it is simply
that doctors are powerless to treat it. In fact, it is generally a weaker form
of the pathogen.1

Is Wieland correct in this statement?
Well yes, he is generally correct - sort of.Most resistant strains of infectious bacteria are not "more virulent" outside of their ability to resist certain antibiotics.Often they are even less metabolically active and less able to cause
disease than their wild-type counterparts, at least for a while, but not always
- and not even if they were hindered initially since they may be able to
compensate for initial losses of function with compensatory mutations.48Besides, this whole argument says nothing about an organism's ability to
evolve new functions.Just because new functions
evolved in a weaker organism or even if the new functions caused the organism to
be "weak" does not negate the fact that a new function evolved that was not
there before.

On top of this, many new functions, that are
known to be the result of spontaneous genetic mutations, are not harmful in the
least, but are entirely helpful.For example, consider again Barry Hall's work with lactase
evolution in E. coli.The evolution of the
lactase function in these colonies of E. coli came at no detrimental cost
as compared to the mutant wild-type strains of K12 E. coli
used by Hall.9

There are many other such examples in literature
(such as the well known example of nylonase evolution).
However, what is especially interesting to me is that the evolution of new
functions generally involves just one or maybe two point mutations in functional
sequences that are relatively short. Functions of greater complexity,
requiring anything more than a few hundred fairly specified amino acid residues
working together at the same time, simply do not evolve. Specifically,
multi-protein systems, like bacterial systems of motility (i.e., the flagellum,
with more than 20 different required parts and well over 5,000 fairly specified
residues at minimum), where all the uniquely interdependent protein parts work
together at the same time in a specified orientation with each other, just do
not evolve in real life. Not even a single proposed step in flagellar
evolution scenarios has ever been shown to evolve in real life - not one! Other
examples of such cellular functions include DNA transcription and mRNA
translation, phagocytosis, endocytosis and pinocytosis (cell "eating" and
"drinking"), and many more.

But what about diseases such as sickle-cell
anemia and various forms of cancer? Are these really examples of the
evolution of new functions?I would say that they are, but does this evolution explain the theory of
common descent when one moves up the ladder of functional complexity?Consider the following comments from Dr. Felix Konotey-Ahulu, an
authority on sickle-cell anemia:

Sixth-graders I have lectured on
genetic counseling invariably pop some questions such as: 'Is it true that the
sickle-cell phenomenon has established Darwinian evolution as fact?' Behind the
question, of course, lies the assumption that observing selection/adaptation
involving a mutation (an inherited random change or defect) somehow implies that
the more complicated forms seen today arose from simpler forms traced ultimately
to one-cell organisms.7

So why, if new functions can and do evolve,
can't Darwinian evolution explain the origin of the huge variety of life forms
that we see in the natural world?It seems rather intuitive that if small functionally unique changes can
occur that these changes should be able to add up over time to produce larger
and still larger changes - until the diversity and complexity that we see around
us comes into being.Where then is the limitation to
such evolution?

Over and over again we see that rapid evolution
of new function is always the result of one or rarely two point mutations to
previously existing genetic elements.It is obvious then that the evolution of new functions or traits does
happen via such mutations combined with the power of natural selection.This cannot be denied nor should we find it surprising.It is statistically likely, given that a gene is but one point mutation
away from new function, that such a gene will chance upon such a beneficial
target sequence that is no more than an arm's distance away in a reasonable
amount of time. The problem for evolution comes when
more than a single point mutation is needed to achieve a given function - such
as appears to be the case with the evolution of the penicillinase and other such
relatively simple single-protein enzymatic-type functions.

Even the evolution of the beta-galactosidase
function in the E. coli mutants studied by Hall required the prior
existence of the evolved beta-galactosidase gene (ebg).Colonies of E. coli that had the original lacZ as well as ebgA
genes removed, never evolved b-galactosidase function despite very large
colonies with high mutation rates living in a lactose enriched environment for
tens of thousands of generations.It appears that
without these two genes, no other genes or genetic elements in E. coli
are close enough to the minimum part requirement of beta-galactosidases to
achieve beta-galactosidase activity with just one or two point mutations.
The ratio of what will work compared with what will not work is relatively
small. This small ratio between non-junk and junk creates a neutral gap
between various non-junk functions at a certain level of functional complexity.

For all cellular functions there is a minimum
part requirement consisting of amino acids in specific sequences.
If changed beyond this minimum requirement, all function is lost. All genes and all proteins are in fact, "irreducibly
complex." Despite the fact that many genes and proteins are quite flexible in their
sequencing, all of them have a limit beyond which all beneficial function is
lost.These limits may overlap with other genes and
proteins, in which case, evolution or change between two functional genes or
proteins is possible in a relatively rapid manner.However, as the level of functional complexity increases, the average
neutral gaps between potentially beneficial proteins also increase. With
the increase in neutral gaps comes a decrease in functional overlap between
various sequences. At this point,multiple neutral
mutations are required before a new beneficial function can be realized. These
multiple mutations are invisible to the powers of natural selection. That
is why such changes are called "neutral". They are neutral with respect to
functional change and this makes them neutral with respect to any selective
advantage that nature might provide.

So, the traversing of such a gap
requires a truly random walk. And, as we all know, it is much faster to go
from point A to point B by following a straight line. Walking along a
random curvy path will take a whole lot longer. This is what happens with
evolution when the pathway is neutral with regard to any sequentially selective
advantages. The evolution of new functions at such levels requires
exponentially greater amounts of time.

Clearly then, these neutral gaps
present insurmountable blockades to the evolution of new functions beyond the
lowest levels of functional complexity - even for such large populations and
such rapid generation turnovers as are realized in bacterial colonies (and we
are talking trillions upon trillions of years for the crossing of neutral gaps
averaging no more than a couple dozen
residue changes wide).Since such
isolated functions of higher and higher levels of complexity do in fact exist in
the natural world, it seems extremely difficult for the theory of evolution or
any other purely naturalistic theory based on mindless naturalistic processes to
explain their existence outside of deliberate design.

This is probably the most common mechanism of
antibiotic action.When an antibiotic binds to its target, it limits the target's ability to
perform its normal function.So, if a mutation
occurred that blocked that antibiotic's ability to bind to the target, the
antibiotic would loose its ability to hinder the function of that target.
The actions of many types of antibiotics are successfully prevented by such
mutations.

Aminoglycosides and macrolides are antibiotics
that target bacterial protein synthesis.The great differences between prokaryotic and eukaryotic (bacteria and
humans) systems of protein synthesis allows for their great specificity in the
targeting of bacteria without harming the human host.
In order to understand this process more clearly we must first review the
process of protein formation from the coded information found in a bacterium's
DNA (deoxyribonucleic acid).

Of course the information in DNA is stored in
coded form by the linear sequence of the chemical "letters" labeled with the
names adenine (A), thymine (T), guanine (G), and cytosine (C).
These chemical letters are defined according to three-letter sequences or
"words" called "codons." Each one of these codons are defined by
the "genetic code" of the bacterium as representing one of twenty amino acids
(amino acids are the basic building blocks of proteins).10

This system works very much like the Morse Code.
Each symbol in the Morse Code represents one of the twenty-six letters in the
alphabet.In just the same way, codons represent the "letters" or amino acids
in the protein "alphabet." However, DNA does not get
"translated" into proteins directly.DNA is used as a template or a "master copy".There is another molecule called, "messenger RNA" (ribonucleic acid),
that is used as a "working copy" of the DNA.The cell in fact "transcribes" the desired portion of DNA into mRNA and
then the mRNA is "translated" into appropriate amino acid sequence or protein
molecule.RNA molecules are similar to DNA molecules
but do have some important differences.Thymine (T)
in DNA is replaced by uracil (U) in RNA.10
RNA is also a single stranded molecule while DNA is double stranded. In any case, it is the mRNA strand that is translated into an
amino acid / protein sequence. The process of protein translation is quite complicated, but
basically it involves the recognition of another molecule called, "transfer
RNA" (tRNA).tRNA has a two-dimensional
shape that resembles a cross (note illustration).One of the three loops that make up the arms of the cross
is called the "anticodon loop." This loop contains
a three-letter sequence that matches or compliments a specific codon on the mRNA
molecule.For example, in the figure of mRNA
(pictured above) "Codon 1" reads, "GCU." The matching anticodon of tRNA would read, "CGA."
This matching of codon and anticodon is specifically linked to a particular
amino acid. Also, each tRNA has a "3'-end" that is called, "the site of
amino acid attachment." The association of a
particular amino acid with a particular tRNA and its anticodon is extremely
specific.Therefore, a particular anticodon is
always associated with the same amino acid. 12 However, in order to
bring the codon and anticodon together in such a way where amino acid linking
can be made in sequential order, different molecules of RNA called "ribosomes"
(rRNA)
are needed. Ribosomes consist of two main parts or "subunits." These subunits come together around the mRNA like two sides of a hotdog bun
surrounding the mRNA hotdog. In certain bacteria, such as
E. coli, the ribosomal subunits come
together in a specific location on the mRNA chain called the "Shine-Delgarno
sequence" that reads, "AGGAGG." This particular sequence is located just
in front of another sequence known as the "start" sequence or "AUG" sequence.
Without the Shine-Delgarno sequence, the ribosomes would not know where to begin
the process of protein sequencing. Obviously if the sequencing started in
the middle of the mRNA molecule, one half of a desired protein would be made.
So, it is vital that the process be able to recognize were to start sequencing.
After the "beginning" of the mRNA molecule is recognized, the beginning
message is recognized by a special "initiator tRNA." The initiator tRNA
always has a methionine amino acid attached to it that will form the
"N-terminal" amino acid in the growing peptide chain. This methionine
molecule may be removed however before the functional protein has been
completed. The process so far described is called, "initiation." The
next required step is called, "elongation."

Elongation of the peptide chain
involves the addition of amino acids to the carboxyl end of the growing
polypeptide chain.During elongation the ribosomal
complex moves from the 5'-end to the 3'-end of the mRNA molecule that is being
translated.Elongation is made possible by the fact
that the ribosomal complex has two binding sites for tRNA molecules called the
"A" and the "P" sites that involve both ribosomal subunits.
The A and P sites cover two neighboring codons on the mRNA chain.
During translation the A site binds an incoming tRNA that matches the mRNA codon that
is currently occupying the A site.This codon
specifies the next amino acid to be added to the growing polypeptide chain.
The P site is occupied by a tRNA molecule that matches the codon that was at the
A site just one step before.In other words, as the
ribosome complex moves along the mRNA chain, the codons come in one side of the
ribosome complex and leave out the other side.The incoming codons pass sequentially through the A site and then the P
site before leaving the ribosomal complex.As the complex moves from one codon to the next, the growing amino acid
chain on the tRNA occupying the P site is attached to the N-terminal end of the
single amino acid occupying the A site.After this is done, the complex moves forward to the next
codon on the mRNA.As the complex moves forward, the
tRNA molecule that was occupying the P site is bumped out if its place by the
tRNA that was occupying the A site.Now the former A site tRNA occupant is the new P
site occupant, still with the growing polypeptide chain attached to it.
Then, the next tRNA molecule that matches the next codon arrives and the whole
process repeats itself over and over again until the entire polypeptide is
formed.12

Many more molecules and interactions are needed
and four of the steps require energy in the form of energy molecules called
adenosine triphosphate (ATP) and guanosine triphosphate (GTP).
In fact, the addition of one amino acid to a growing peptide chain requires the
use of 2 ATPs and 2 GTPs.12Even from
this short and highly simplified overview of polypeptide synthesis, it is clear
that it is a very complex, detail dependent, and highly specific process.
It is similar to very finely tuned assembly line.
Obviously then, if any one step is blocked the entire process would fail and the
host cell would quickly die.

Many antibiotics work by blocking one step or
another in the process of protein production.Aminoglycosides, such as kenamycin and streptomycin, interfere with
protein production by binding to the smaller of the two ribosomal subunits (30S
subunit).This is significant because within
the ribosomal structure translational accuracy requires that the anti-codon of
tRNA and the codon of mRNA be specifically recognized.
This recognition occurs with the 30S ribosomal subunit.
Even slight changes in this region of the 30S ribosomal subunit interfere with
the fidelity of this process of recognition.The
requirement for such high fidelity in this step creates a weak point in the
process.

Aminoglycosides have been extremely successful
in exploiting this weakness. For example, the
aminoglycoside "streptomycin" specifically attaches to a region around
the " site" of the 30S ribosomal subunit (specifically the 16S sub-subunit)
along with some other proteins within the 30S subunit and stabilizes a
conformational state in which the initial binding of "non-cognate" or
non-similar tRNAs are favored.This results in coding errors and, of course, the
production of nonfunctional proteins.Even today
aminoglycosides remain useful for the treatment of infections caused by aerobic
Gram-negative bacteria.Also, both streptomycin and
kanamycin are important second-line chemotherapeutics in the treatment of
multi-drug resistant tuberculosis.13

It is evident then that any change that affects
the binding of aminoglycoside antibiotics to this 30S ribosomal subunit would
block their adverse effects on protein synthesis, thus giving the bacterium
"resistance" to this type of antibiotic.It is not
too surprising then that streptomycin resistance involves a single point
mutation in the gene (rrs locus) that codes for the 16S
sub-subunit of the ribosome complex.Another point
mutation in a gene (rpsL gene) that codes for the protein
portion (S12 ribosomal protein) of this subunit can also result in streptomycin
resistance.14Both of these mutations
cause a decreased binding affinity of streptomycin with its normal binding site
on the 16S sub-subunit.

Obviously then, those bacteria with fewer copies
of the ribosomal genes would achieve resistance much faster than those having
multiple copies of these genes.This is exactly what happens.For example,
M. tuberculosis only has a single copy of the rRNA genes and yet it can go
from a streptomycin resistant rate of 2% to over 80% within three or four months
time (if streptomycin is the sole antibiotic of use).15
However, E. coli, although much faster growing than M. tuberculosis, are
far less likely to develop resistance because they harbor multiple copies of the
rRNA genes.Multiple copies of genes and a rapid
growth rate means that multiple independent mutations are needed to prevent
streptomycin susceptibility.When two or more independent point mutations are required
for resistance, this takes much more time to achieve than a single mutational
event that instantly results in streptomycin resistance.

Macrolide antibiotics, such as erythromycin, are
similar to aminoglycosides in that their target activity involves the ribosomal
complex.However, instead of attacking the 30S
subunit, they attack the larger 50S subunit (specifically at the peptidyl
transferase center in the 23S sub-subunit).Since
erythromycin is produced naturally by bacteria such as Streptomyces
erythreus, it is no surprise then that a specific gene
also exists within various bacteria that inhibits erythromycin function.
This gene is known as the "erythromycin resistance methylase gene" or "Erm" gene.This gene either mono- or di-methylates "adenine 2058" in
the peptidyl transferase loop of the rRNA.17This methylation blocks the erythromycin affinity and
restores the fidelity of the ribosome.Erythromycin resistance that involves the Erm gene does not occur through
point mutation, but through an isolation of those bacteria that previously had
the Erm gene present in their gene pool.However, even without the Erm gene, all is not lost for
bacterial colonies under macrolide attack.Macrolide resistance can be also achieved by a point mutation that
involves that replacement of an adenine molecule at position 2058 with either
guanosine (G), cytosine (C), or uracil (U).The replacement of adenine at two other locations (G2057A and A2059G) is
also known to result in macrolide resistance. 16

So, macrolide resistance can be achieved either
through the previous existence of the Erm gene or through a spontaneous point
mutation to three different sites within the 23S sub-subunit of the ribosomal
complex.As would seem intuitive, the Erm gene and
its variants are found in rapidly growing bacteria, such as Staphylococcus
and Streptococcus, while spontaneous mutations of the 2058 site and the
two other related sites usually occurs in species that contain a small number of
23S rRNA genes (such as Mycobacterium).As
with aminoglycoside resistance, rapidly growing bacteria require higher levels
of protein production and thus need more rapid protection of most of their rRNA
genes and gene products.Random point mutations take
too much time to build up in a population with such needs.
However, slower growing bacteria with few copies of rRNA genes can achieve full
resistance with just one point mutation.18

Like protein synthesis, DNA replication and
repair is extraordinarily complicated.However, presented here are a few basic steps that will give a general
idea of the processes involved, how antibiotics can interfere with these
processes, and how bacteria can evolve resistance to the action of these
antibiotics.

The first step in DNA
synthesis or replication involves the separation of the two strands in the DNA
double helix.Only when separated can each of the
individual strands begin the replication process.
The reason for this is because the replicating enzymes (polymerases) use only
single-stranded DNA as a template.
In prokaryotes, such as bacteria, DNA replication can only begin at a single
discrete site called the, "origin of replication."
As the two strands of DNA are unwound and are separated from each other, they
form a "V" shape where the active replication or copying of the two strands of
DNA occurs.This area is called the, "replication
fork."This fork moves along the DNA molecule as
synthesis occurs in both directions away from the origin of replication.12

A special group of proteins is
responsible for maintaining the separation of the DNA strands and for unwinding
the double helix ahead of the advancing replication fork.These include the helix-destabilizing (HD) proteins (also
called single-stranded DNA binding proteins or SSBs) and DNA helix-unwinding
proteins (also called helicases).HD proteins bind to single-stranded DNA in a cooperative manner.That is, the
binding of one molecule makes it easier for additional molecules of HD protein
to bind.These proteins serve a dual function in
that they not only keep the two strands of DNA separated during replication, but
also protect the single-stranded DNA from cleavage by nucleases that break apart
unprotected segments of single-stranded DNA.The DNA
helicases bind to single-stranded DNA near the replication fork and then move
into the neighboring double-stranded region, forcing the strands apart.These enzymes require energy in the form of ATP.
Once the strands have been separated by the helicases they are then stabilized
by the HD proteins.12

The act of separating the strands of DNA causes
a "supercoiling" tension downstream from the replication fork. Unchecked,
this tension would quickly increased and eventually prevent further progression
of the replication fork. However, another group of enzymes, the topoisomerases introduce "swivel" points along the double helix to release the "supercoiling" tension caused by the uncoiling of the helical DNA.This supercoiling phenomenon can be demonstrated by tying two ends of a
rope together, twisting them to form a double-helix, and then pulling them apart
in the center.The twists in the rope on either side
of the separation will twist more and more until they greatly limit further
separation of the two ropes in the middle.DNA
topoisomerases come in different types. Type I topoisomerases, also
called DNA swivelases, reversibly cuts a single
strand of the double helix thereby allowing the cut strand to swivel around the
uncut strand to relieve the built up tension on the two strands.
After the swiveling has released the tension, the swivelase enzymes can reseal
the cut.Therefore, swivelases have both nuclease (strand-cutting)
as well as ligase (strand-resealing) activities.They do not require ATP but rather store energy from the
phosphodiester bond that they cleaved and use the stored energy to reseal the
strand.Type II topoisomerases, also called DNA
gyrases, bind tightly to both strands of the DNA and make transient breaks in
both strands of the DNA helix.The enzyme then
causes a second stretch of the DNA double-helix to pass through the break and
finally reseals the break.The result is a "negative
supertwist" that allows easier unwinding of the DNA double-helix.12

Once the strands are separated, both
of the original or parental strands of DNA serve as a template for the synthesis
of compliment strands of DNA.The synthesis of each of these new strands of DNA is the result of DNA
polymerases.There is a limitation to how these
polymerases read however.Just as the English
language is read only from left to right, DNA polymerases can only "read"
DNA strands from the 3' to the 5' direction (This is can get confusing, but
remember that the polymerases read DNA in the 3' to 5' direction, but
form DNA in the 5' to 3'
direction).Therefore, beginning with one parental
double-helix, the two growing nucleotide chains must grow in opposite directions
- one in the 5' to 3' direction toward the replication fork, and the other one
in the 5' to 3' direction away from the replication fork.
This fairly complicated ability is accomplished by a slightly different
mechanism for each strand.The strand that is being
copied in the direction of the advancing replication fork is called the "leading
strand." Since the copying for this strand is in
the same direction of the advancing replication fork, the synthesis of DNA is
not interrupted, but occurs continuously.However, the other strand, the "lagging strand," is not
so lucky since it must grow away from the direction of the replication fork.
It is synthesized discontinuously, with small fragments of DNA being copied near
the replication fork.These short stretches of
discontinuous DNA, termed "Okazaki fragments," are eventually joined to become a
single, continuous strand.However, these are not
the only problems that must be overcome by the DNA polymerases.12

Before DNA polymerases can copy DNA, they must be initiated by an
RNA polymerase or "primer" called primase.Interestingly enough they cannot initiate DNA synthesis by themselves.
The primase primer synthesizes short stretches of RNA (approximately ten
nucleotides in length) that are complementary to the DNA template.
As seen in the figure, these short RNA sequences are constantly being
synthesized on the lagging strand, but relatively few are required for the
leading strand.But even before primase can bind to
DNA a "prepriming complex" must bind the single stranded DNA first and displace
some of the single-stranded DNA binding proteins.
Only after the prepriming complex binds can the primase bind.
The prepriming complex plus the RNA primase is called the "primosome." 12

Once the primosome is formed on the parent
strand of DNA, DNA chain elongation can begin via the catalytic activity of DNA
polymerase III.DNA polymerase III first recognizes
the 3' hydroxyl group of the RNA primer as the acceptor of the first
deoxyribonucleotide.Of course, from this starting
point DNA chain synthesis proceeds in the 5' to 3' direction "antiparallel" to
the parental strand, which is read in the 3' to the 5' direction.
All four deoxyribonuceoside triphosphates (dATP, dTTP, dCTP, and dGTP) must be
present in sufficient quantities for DNA elongation to occur.If one of the four is in short supply, DNA synthesis will
fail.

Consider also the importance of the 3' hydroxyl
group on the deoxyribose ring.This 3' hydroxyl
group forms a phosphodiester bond with the next nucleotide at its 5' phosphate
group.If a nucleotide with an absent 3' hydroxyl
group is added to a growing DNA chain, further elongation cannot continue.
Certain chemotherapeutic (cytosine arabinoside-araC) and antiviral (araA) agents
take advantage of this fact to slow the division of rapidly growing cancer cells
and viruses.12

Of course, it gets even more complicated.
In order to prevent as many copying errors as possible from making it to the
next generation, DNA polymerase III also has a "proofreading" function that
works in the 3' to 5' direction (3' to 5' exonuclease).
As each nucleotide is added to the chain, DNA polymerase III checks to make sure
that the added nucleotide is, in fact, correctly matched to its complementary
base on the template and edits its mistakes.For example, if the template base is adenine and the
enzyme mistakenly inserts a cytosine instead of a thymine in the new chain, DNA
polymerase III hydrolytically removes the misplaced cytosine and replaces it
with thymine.This exonuclease activity is specific
in that it only replaces mismatched nucleotides but never correctly paired
nucleotides.12

DNA polymerase III continues to synthesize DNA
until it is blocked by a stretch of RNA primer.When this occurs, the RNA is excised and the gap if
filled by a separate DNA polymerase by the name of DNA polymerase I.
In addition to all of the abilities of DNA polymerase III, DNA polymerase I has
an exonuclease activity that removes the sections of RNA primer.
In order to do this, DNA polymerase I first recognizes the space or "nick"
between the 3'-end of the newly synthesized DNA strand and the 5'-end of the
adjacent RNA primer.The final linkage
between the 5'-phosphate group on the DNA chain that was synthesized by DNA
polymerase III and the 3'-hydroxyl group on the chain made by DNA polymerase I
is catalyzed by another enzyme, DNA ligase, plus one molecule of ATP. 12

The surrounding environment is constantly
damaging DNA.The damaging agent is usually chemical, but radiation is also a
significant mutagen.Sometimes spontaneous changes
also occur that are unrelated to any other agent.Such changes or mutations are usually neutral or harmful."Beneficial" mutations are extremely rare.So, it is fortunate that cells are remarkably efficient at damage
control.Most of these repair mechanisms involve
recognizing the lesion, removing the damaged section of DNA, and, using the
sister strand of DNA as a template, filling in the gap left by the excision of
the abnormal section of DNA.

Ultraviolet light specifically causes the
covalent joining of two adjacent pyrimidines (usually thymines), producing a "pyrimidine
dimer."These dimers prevent DNA polymerase from replicating DNA beyond the site
of the dimer.In order to remove these dimers a
specialized UV-specific endonuclease recognizes the dimer and cleaves the
damaged strand on the 5' side of the dimer.Next, an excision exonuclease recognizes the incision made by the
endonuclease.In E. coli, this 5' to 3'
exonuclease activity is associated with DNA polymerase I.The gap left by the removal of the thymine dimer is filled, using the
sister strand as a template, by DNA polymerase I.And, as with normal DNA synthesis, the last phosphodiester bond is formed
with the help of DNA ligase.Without this repair
mechanism in place, dimers build up in cells, rapidly resulting in cancers such
as skin cancer.Those with increased rates of such
cancers commonly have a problem with UV-specific endonuclease activity.12

Other forms of damage to DNA occur
spontaneously.For example, over time cytosine slowly looses its amino group.When this happens, it is no longer cytosine, but uracil.Other types of damage can be caused by chemical interactions with DNA.For example, nitrous acid (formed by the cell from precursors such as
nitrosamines, nitrites, and nitrates), removes the amino group from cytosine,
adenine, and guanine bases.Bases may also be
completely lost spontaneously.For example, around 10,000 purine bases are lost spontaneously per day in
an average cell.DNA lesions involving alteration or
loss must be corrected rapidly in order to keep up with the rapid rate at which
they occur.And in fact, this is what happens.
Abnormal bases are recognized by specific glycosidases that hydrolytically
cleave the base out of the strand.Specific
endonucleases recognize that a base is missing and fill in the gap as usual.In addition, other endonucleases recognize abnormal bases and initiate
the nucleotide excision without first requiring that the base be cleaved from
its sugar backbone by a specific glycosidase.12

So, vital DNA repair is accomplished by a large
number of unique and highly specific enzymes working in concert.
Their importance is quickly realized if even one or two of them are lost,
because sickness or even cell death quickly follows.

Fluoroquinolones (ie: ciprofloxacin) are
synthetic antibiotics that target both DNA gyrases as well as topoisomerases
(type "IV").The gyrases are heterotetramers (A2B2) whose subunits are encoded by the gyrA
and gyrB genes.Topoisomerase IV is also a heterotetramer composed of distinct subunits
called ParC and ParE.ParC is a homologue of gyrA
while ParE matches gyrB.Specifically, topoisomerase IV is the main enzyme that removes the
linking of daughter chromosomes after a round of DNA replication (allows
cellular segregation to occur).19

Fluoroquinolones specifically trap doubly
cleaved DNA on the gyrase structure.Double-stranded breaks accumulate as a consequence since the gyrase
enzymes are now incapable of reattaching the broken strands.This blocks the progress of the replication fork and the cells dies.Fluoroquinolones also stabilize the DNA-topoisomerase IV complex, thus
hindering chromosomal separation.19,20Interestingly enough, gyrases are the primary target in Gram-negative
bacteria while topoisomerase IV is the primary target in Gram-positive bacteria.19,21

In Gram-positive bacterial species
fluoroquinolone resistance is the result of a single point mutation (although
several different point mutations between "residues 67-106" can result in quinolone resistance). The beneficial point mutations generally involve
the "quinolone resistance-determining region" (QRDR) of the A subunit of DNA gyrase.The QRDR is located in the N-terminal region of the gyrase protein.This region is located on a specific area of the gyrase enzyme where it
bends or distorts the strand of DNA that is cleaved.It is thought that mutations to this area reduce the probability of
enzyme-quinolone-DNA complex formation.A mutation
in this area, that results in just one amino acid change, can cause a 4 to
8-fold increase in fluoroquinolone resistance.20,21The theory put forward to explain Gram-negative resistance suggests that
specific mutations could change the stability of the topoisomerase-DNA-quinolone
complex without affecting the probability of its formation.22

As briefly touched on earlier, proteins are not
made directly from the DNA or "master copy." Instead,
"working copies" are needed in the form of messenger RNA (mRNA) molecules.The process of making mRNA from DNA is called transcription while the
process of making proteins from mRNA is called translation.10

A central feature of transcription is that the
process of selecting regions of DNA to copy into mRNA is highly specific.
This specificity is primarily due to "signals" that are embedded in nucleotide
sequences of the DNA.These signals instruct the RNA
polymerase where to start transcription, how often to start transcription, and
where to stop transcription.These signals are extremely important because the biochemical
differentiation of different cellular structures and ultimately tissues and
organ systems is due to the selectivity of this transcription process.10

A second important feature of transcription is
that the mRNA transcripts, which initially are faithful copies of the DNA,
undergo various modifications (terminal additions, base modifications, trimming,
internal segment removal, and splicing) that convert the inactive primary
transcript into a functional molecule.10

In bacteria RNA polymerase there is a
multi-subunit enzyme that recognizes specific nucleotide sequences such as the
â€œpromoter regionâ€ at the beginning of a stretch of DNA.
After it makes a complementary RNA copy of the DNA template, the polymerase
enzyme also recognizes the end or "terminator region" of the DNA sequence that
is to be transcribed.Four of the RNA polymerase
subunits (2a, 1b, and 1b' ) are responsible for the 5' to 3' RNA polymerase
activity.However this core enzyme complex cannot
recognize the promoter region of the DNA template by itself.
This recognition requires the action of the "s-subunit" or "s-factor" that
enables the polymerase to recognize the promoter regions on the DNA.As far as termination recognition goes, some termination regions are
recognized by the RNA polymerase direction while others require yet another
subunit called the "r-factor" (in E. coli).12

So, there are three basic steps involved in RNA
synthesis.These are initiation, elongation and termination.12Consider the following illustration of these steps:

As previously mentioned, the initiation of
transcription involves the binding of RNA polymerase to a promoter region on the
DNA.Certain specific promoter sequences are known.One such sequence is called the, "Pribnow box." This is a sequence of seven nucleotides centered around ten bases
upstream from the initial base of the mRNA coding sequence.A second nucleotide sequence, "TGTTG" is also recognized by RNA
polymerase.This sequence is located about 35 bases
upstream.Of course this is based on a prokaryotic
model.The structure of eukaryotic promoters is much more complex than this.12

Once the promoter is recognized the RNA
polymerase begins to synthesize the RNA transcript.
Unlike DNA polymerase, RNA polymerase does not require a primer and has no known
endonuclease or exonuclease activities. The binding of RNA polymerase to the
double-stranded DNA template results in a local unwinding of the DNA helix.Therefore, RNA polymerase does not require additional unwinding enzymes
or helix destabilizing enzymes as does DNA polymerase.Like DNA polymerase, RNA polymerase uses nucleoside triphosphates and
releases a molecule of pyrophosphate each time a nucleotide is added to the
growing chain.The growing RNA molecule is
synthesized from its 5'-end to its 3'-end, antiparallel to its DNA template (The
only difference between the nucleotides used in DNA and RNA is the substitution
of a uracil (U) in RNA for a thymine (T) in DNA).The process of elongation continues until a termination signal is
reached.12

RNA polymerase can sometimes recognize termination regions all by itself.However, as previously mentioned, the r-factor may be required for the
release of the RNA product as well as the RNA polymerase.As the figure illustrates, the termination region of the DNA exhibits a
twofold symmetry owing to the presence of a palindrome.The RNA transcript of the DNA palindrome forms a stable "hairpin turn"
that slows down the progress of the RNA polymerase at this terminator site.
Meanwhile, the r-factor binds to the 5'-end of the RNA transcript and moves
along the newly forming RNA molecule, following the polymerase at some distance
as it forms the RNA. When the polymerase pauses at the terminator site, the
r-factor catches up to the polymerase and causes termination.12

After termination and some post-transcriptional modifications, the mRNA is
ready to be translated into protein. rRNA and tRNA transcripts are made in
the same way.Long precursor transcripts are formed that contain all the subunit RNA
sections.Each of these subunits is cut out of the
precursor molecule and modified before they become active rRNA and tRNA
molecules.12

Rifampin, a semi-synthetic derivative of
rifamycin, is an important antibiotic in the treatment of mycobacterial
infections (i.e., Tuberculosis).Rifampin specifically targets the b-subunit of RNA polymerase.The formation of a drug-enzyme complex inhibits the initiation of chain
formation in the process of RNA synthesis.Interestingly, the existence of rifamycin-inactivating enzymes has not
been reported (an unusual event for drugs which are natural products).Thus, antibiotic resistance mechanisms rely solely on mutagenesis of the
Rifampin target.A single mutation to this target
results in resistance.Obviously then, susceptible
organisms, such as mycobacteria, can develop rifampin resistance at incredibly
rapid rates.14,15,23Specific insertions and deletion mutations of bacterial RNA polymerase
usually occur within 3 short, highly conserved regions of the b subunit, and
encompass the "hot spot" area between residues 505 and 534 (E. coli
numbering).23New synthetic derivatives
such as rifabutin, rifapentine, and KRM-1648 display strong cross-resistance
with rifampin and also are not any more effective than rifampin.14,24
So, although rifampin is highly effective against M. tuberculosis
infections, it should always be used with at least one other antibiotic.

Actinomycin D also acts by interfering with RNA
synthesis by binding to the DNA template and interfering with the movement of
RNA polymerase along the DNA.12I am not aware of a target mechanism of resistance to actinomycin D.

In a bacterium the "cell
wall" is the outermost protective covering (as is illustrated in the figure).
Almost all bacteria have cell walls except for a few, such as the Mycoplasma
species, which are bound by thin cell membrane instead of an actual wall.11 The cell wall is a multi-layered structure located
externally to the cytoplasmic membrane.It is
composed of an inner layer of peptidoglycan surrounded by an outer membrane that
varies in thickness and chemical composition depending upon the bacterial type.The peptidoglycan layer provides structural support and maintains the
characteristic shape of the cell.11

The structure, chemical composition, and
thickness of the cell wall differ in gram-positive and gram-negative bacteria.
Perhaps the most obvious difference is the dramatically thicker peptidoglycan
layer of gram-positive bacteria.Some gram-positive
bacteria also have a layer of teichoic acid outside the peptidoglycan layer
whereas gram-negative bacteria do not.There are many other key differences and important features between
gram-positive and gram-negative bacteria, but since antibiotics primarily target
the peptidoglycan layer, we will concern ourselves primarily with its structural
formation.

Peptidoglycan is a complex,
interwoven network of sugars and proteins that surrounds the entire cell wall.
It is found only in bacterial cell walls.It
provides rigid support for the cell, is important in maintaining the
characteristic shape of the cell, and allows the cell to withstand environments
with low osmotic pressure - such as pure water.

The term, "peptidoglycan" is derived from the
peptides and the sugars (glycans) that make up the molecule.
The peptidoglycan molecule has a carbohydrate backbone composed of alternating
N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) molecules.Attached to each of the muramic acid molecules is a tetrapeptide
consisting of both D- and L-amino acids - the precise composition of which
differs from one bacterium to another.

Two of these amino acids, diaminopimelic acid
and D-alanine, are worthy of special attention.
Diaminopimelic acid is unique to bacterial cell walls and D-alanine is involved
in the cross linking of the
tetrapeptides (and is specifically targeted by penicillin type antibiotics).
Note also that this tetrapeptide contains rare D-isomers of amino acids (most
proteins contain the L-isomer). Also, since peptidoglycan is present in
bacteria but not in humans, it is an excellent target for antibiotics.
Without a functional peptidoglycan layer, the bacteria swell due to osmotic
pressures and rupture in low osmotic environments.11

The biosynthesis of
peptidoglycan involves a number of cytoplasmic steps, after which the
disaccharide-pentapeptide intermediate is attached to a lipid carrier and is
translocated across the membrane into the bacterial periplasm (The space between
the membrane and the cell wall).In the periplasm,
the glycosyl transfer and transpeptidation activities of "penicillin-binding
proteins" (PBPs), or "transpeptidases", complete the polymerization of the
sugar backbone as well as the cross-linking of the pentapeptide chains (or "stem
peptides"). 11

Beta-lactam antibiotics (penicillins, cephalosporins, etc.) kill
gram-positive bacteria by inhibiting PBPs - the enzymes that catalyze the final
cross-linking steps in the synthesis of peptidoglycan.25For example, in Staphylococcus aureus, transpeptidation occurs
between the amino group on the end of the pentaglycine cross-link and the
terminal carboxyl group of the D-alanine on the tetrapeptide side chain.Since the structure and chemical nature of
penicillin is similar to that of the dipeptide D-alanyl-D-alanine,
penicillin can bind to the active site of the transpeptidases as a "pseudosubstrate"
and inhibit their activity.26The PBP
active sites, once acylated by penicillin, deacylate very slowly and are
subsequently incapable of performing regular cross-linking functions. Since PBPs
are the enzymes that link the sugar backbone together as well as the cross-links
of the protein "stems" that hold the sugar chains together, anything that
interferes with their action would result in a loss of cell peptidoglycan
integrity. A second factor that comes into play concerns the action of
peptidoglycan hydrolases or "murein hydrolases" (murein is a synonym for
peptidoglycan).Murein hydrolases degrade peptidoglycan and are naturally produced by
many types of bacteria for the purpose of peptidoglycan upkeep and remodeling.However, if the action of these hydrolases is limited in some way, the
integrity of the cell wall will remain intact a great deal longer even in the
presence of antibiotics that destroy the building functions of PBPs. For example, if a wall cannot be torn down very easily, it is
not as important that one be able to repair wall damage quickly.In fact, some bacteria, such as S. aureus, are tolerant to the
action of penicillins due to the fact that autolytic murein hydrolases are not
activated.11A "tolerant" organism is
one that is inhibited, but not killed by a usually bactericidal antibiotic.

When one thinks of penicillin-type antibiotic
resistance, the enzyme "penicillinase" often comes to mind.
However, there are several other ways that bacteria become resistant to
penicillin.A notable example is occurs S. pneumoniae.beta-lactamases have never been identified in S. pneumoniae.
And yet, these bacteria are capable of penicillin resistance due to modification
of their PBPs.27,28,29Many
specific point mutations have been noted to confer penicillin resistance, but
all of them are located in the transpeptidase domain - with high recurrence at
locations "338 and 552". "Thr 338" is immediately
adjacent to the catalytic "Ser 337" and is buried in a small cavity shielded
from the active site.30,31 This cavity also harbors a buried water
molecule, which is coordinated to the side chain hydroxyl group of Thr 338,
among others. The mutation of Thr 338 to glycine, alanine, proline or valine
lowers the acylation efficiency of PBP for b-lactams, even though this residue
is not in direct contact with the antibiotic.Consequently, in the absence of a crucial hydroxyl group, the buried
water molecule can no longer be stabilized. This reduces the antibiotic
acylation efficiency.32Further
mutagenesis efforts identified that mutation of "Gln 552" into a glutamate
residue caused a severe reduction in acylation efficiency for both cefotaxime
and penicillin G - probably due to electrostatic effects between the incoming
antibiotic and the residue, which borders the active site.33

Another example of PBP-related antibiotic
resistance is detected in methicillin-resistant
S. aureus (MRSA), which is not naturally transformable and
therefore does not develop resistance by increasing gene mosaicity (as occurs in
S. pneumoniae).23In fact, MRSA produces four unmodified PBPs as well as an additional high
molecular weight form (PBP2'), which has low affinity for almost all b-lactam
molecules in use and is not found in normal Gram-positive isolates.In the presence of methicillin, the cell wall of MRSA is altered,
generating a peptidoglycan with fewer oligomeric peptides. This new murein
structure is believed to be a direct consequence of the fact that PBP2' takes
over the regular peptidoglycan transpeptidation functions normally performed by
methicillin-sensitive PBPs.34 The gene coding for PBP2' (mecA)
is found in the same chromosomal location in all MRSA isolates, an observation
which suggests that its introduction occurred only once.23,26

Glycopeptides, such as vancomycin, the famous antibiotic of "last
resort", do not target PBPs, but instead they interfere with peptidoglycan
synthesis by binding to the terminal D-Alanyl-D-Alanine (D-Ala-D-Ala) terminus
of stem peptides.Binding of the glycopeptides to the C-terminal ends of the "stem"
peptides impedes their subsequent recognition by transpeptidases (PBPs).Obviously then, peptidoglycan cross-linking is blocked.In addition, it has also been suggested that glycopeptides block the
transfer of peptidoglycan precursors through the cytoplasmic membrane by steric
hindrance.35This blockade causes a
hindering buildup of peptidoglycan precursors in the cytoplasm of the cell.

The complex between vancomycin and D-Ala-D-Ala
involves a set of five crucial hydrogen bonds between the elongated backbone of
vancomycin and the stem peptide.As would be
expected, vancomycin resistance involves a single structural change in the cell
wall where the D-Ala-D-Ala portion is replaced with D-ala-D-Lactate.This inclusion of an ester instead of an amide bond in the stem peptide
structure profoundly alters its interaction with the vancomycin "backbone"
structure in that there is a loss of one of the five hydrogen bonds.The resulting binding affinity is three orders of magnitude lower than it
was with D-Ala-D-Ala, and bacterocidal activity is lost.36

In several bacterial species, such as in
Enterococcal species (VRE), vancomycin resistance is achieved through the
function of a five-gene operon that is located on a transposon (mobile genetic
element). The mobility of this element suggests that this
resistance mechanism could be acquired by a large number of pathogenic bacteria.The five genes include three structural genes named, vanH, vanA, and van
X.36VanH converts pyruvate to D-lactate.
VanA takes the D-lactate produced by vanH and synthesizes D-Ala-D-Lactate with
it.VanX selectively hydrolyzes all D-Ala-D-Ala peptides that are naturally
produced by the host.Without the availability of
D-Ala-D-Ala, the concentration of D-Ala-D-Lactate builds up in the cell and the
resulting peptidoglycan is made entirely of D-Ala-D-Lactate - given the host
resistance to vancomycin.37Of side interest are the two other proteins
coded by the operon - vanS and vanR.VanS is a
sensor kinase and vanR is a regulator that controls transcription.VanS senses the presence of vancomycin and transmits this message to vanR
via autophosphorylation and phosphoryl transfer.VanR then upregulates the production of D-Ala-D-Lactate. 35,37

In this light, it is interesting to note that
Enterococcal
bacteria do not ever gain vancomycin resistance spontaneously in an isolated
gene pool. The VanHAX genes happen to be on a transposable element in VRE.And, it just so happens that these genes, in the same orientation, are
also found in various bacteria that historically synthesize and secrete
vancomycin. Other types of bacteria, like E. coli also
have very similar genes.38,39Such similarity is thought to represent an evolutionary relationship.

In short, vancomycin resistance did not evolve
in real time.The genes coding for vancomycin resistance were already in the bacterial
gene pool before humans started using vancomycin as an antibiotic. VRE did not
evolve over the course of 15 or so years of vancomycin use in Enterococcal
bacteria.They simply gained vancomycin resistance
via lateral transfer of a pre-formed genetic element that was already there in
the gene pool.
Exactly the same thing happened with penicillin resistance that results from
penicillinase production.The penicillinase gene
does not evolve in real time.It is only inherited via vertical or horizontal transfer - preformed. (Back
to Top)

There is one other cell wall component that we have not yet
discussed which is a very important aspect of mycobacteria.
Mycobacteria have a similar cell wall to gram-positive bacteria, but in
addition, they have an outer leaflet that contains mostly mycolic acids.Mycolic acids are long chain fatty acids that are covalently linked to an
"arabinogalactan" polysaccharide layer.The polysaccharide layer is in turn linked to the underlying
peptidoglycan structure.Mycolic acids provide a
formidable barrier against most antibiotics and are essential for bacterial
viability.11Antibiotics that affect
mycolic acid stability are key to fighting mycobacterial infections.

Antibiotics, such as isoniazid, are thought to
disrupt the biosynthesis of mycolic acids through the inhibition of "inhA," an enzyme (enoyl-ACP reductase) essential in the biosynthesis of long chain
fatty acids and thus mycolic acids.39,40Before isoniazid can act, it must be activated by the
catalase-peroxidase enzyme coded by the KatG gene.Once activated, isoniazid can bind to a target site on the inhA enzyme
and inactivate it.Thus, isoniazid resistance is
often the result of a single point mutation in the inhA gene.This mutation changes the serine occupying position 94 in the inhA enzyme
to an alanine.This change lowers the enzyme's
affinity for a vital NADH molecule by 5 fold.It just so happens that the activated isoniazid molecule covalently
attaches to the NADH molecule that is located within the inhA enzyme's active
site.If the NADH molecule becomes more loosely
attached to this enzyme, the isoniazid antibiotic along with the NADH molecule
fall away from the enzyme and the bacterium gains immunity.This same mutation also gives resistance to isoniazid's structural
analog, ethionamide.40

There are several physical restrictions that
limit antibiotic access to a bacterial target.
Several of these have been mentioned already, to include the thick mycolic acid
layer of protection produced by mycobacteria, and an outer lipid membrane
produced by gram-negative bacteria.11
Such structures limit a large variety of antibiotics from reaching their
targets.For gram-negative bacteria in particular glycopeptides are specifically
limited.Glycopeptides have limited access to their
peptidoglycan precursor targets in gram-negative bacteria because glycopeptides
have large hydrophobic structures in their molecular makeup that cannot readily
cross the gram-negative cell's outer membrane.35

A more complex method of limiting antibiotic
access to target sequences is found in cases of macrolide resistance where the
macrolide antibiotics (like erythromycin and azithromycin) are actually "pumped"
out of the cell.The pump can get rid of the antibiotic faster than it can accumulate in
the cell.One type of pump is produced by the "MefE"
gene.This gene produces a 12-trasmembrane-helix
macromolecule which exports 14 and 15-membered macrolides from the cell - giving
the cell resistance to antibiotics such as erythromycin.
In fact, as many as 85% of erythromycin-resistant S. pneumoniae strains
harbor the MefE gene.18,41

This and other efflux pumps, like the MtrC-MtrD-MtrE system of Neisseria gonorrhoeae
(encoded by the mtrRCDE operon), were already in place, historically, before
humans ever started using azithromycin or erythromycin as antibiotic agents.The MtrCDE pump exports hydrophobic agents, including dyes such as
crystal violet and macrolide antibiotics such as azithromycin and erythromycin.Resistance to these particular antibiotics is the result, not of de novo
efflux pump evolution, but of a loss of regulation of expression of MtrCDE via a
mutation in the repressor (MtrR) region that removes repression or a mutation in
the promoter region of MtrR that decreases promotion of repression (i.e.,
increases production of the protein parts that form the efflux pump).50

Tetracycline resistance is also the result of a
failure of the antibiotic to reach an inhibitory concentration inside the
bacterium.This is due to plasmid-encoded processes that either reduce uptake of the
antibiotic or enhance the antibiotic's transport out of the cell. 11

Likewise, one of two methods of sulfonamide
resistance is mediated by a plasmid-encoded transport system that actively
exports the antibiotic out of the cell.The other mechanism involves a point mutation in the gene coding for the
target enzyme "dihydropteroate synthetase", which reduces the binding affinity
of the antibiotic.11

Quinolone resistance, although often the result
of mutations that modify DNA gyrase, can also be the result of changes in outer
membrane proteins that result in reduced uptake of the antibiotic.11

Perhaps the most famous example of direct
inactivation of an antibiotic by bacteria involves the neutralization of
penicillin and penicillin-like antibiotics via the action of beta-lactamases.
beta-lactamases can be divided into four major groups based on primary structure
alignments, molecular size, and active sites.42,43Class A enzymes harbor a serine in their active site and have an
approximate molecular weight of 30Kda.Also, they
are usually plasmid-encoded and produce the "TEM-1" enzyme.26Class B enzymes are Zn2+-metalloenzymes,
and usually exhibit a broad spectrum of activity.Class C b-lactamases are chromosomally encoded, and, like their class D
counterparts, also harbor a serine in their active sites.Most beta-lactamases produced by gram-positive species are class A
enzymes.25Approximately 90% of all
beta-lactam resistant S. aureus produce beta-lactamases with the
structural and regulatory genes (blaZ, blaI, and blaR1) being harbored by a
plasmid.26

The active sites of class A b-lactamases are
very similar to those of PBPs.This similarity poses a challenge for designing new beta-lactam
antibiotics, which might be less sensitive to beta-lactamase inactivation, but
must still be specifically bound by the PBP active site.34,44 Significant changes to the antibiotic's structure might
avoid inactivation by beta-lactamases, but at the same time it would not be able
to bind to its PBP target site.This creates what
might be called a "win the battle but loose the war" situation.(Back to Top)

Resistance to aminoglycosides is achieved
primarily through chemical modification of the antibiotic.
Three main classes of aminoglycoside-modifying enzymes have been identified in
bacteria.O-phosphotransferases (APHs), which add on phosphate groups; N-acetyl
transfereases (AACs), which acetylate amino groups on the antibiotics, and
nucleotidyl transferases (ANTs), which add "AMP-moieties." 17,45
Antibiotics that have are attached by strategically placed groups of various
kinds no longer have affinity for the target RNA molecule and are thus incapable
of interrupting protein synthesis.

Research done by those
like James R. Knox et al. is commonly used to support the theory that the
penicillinase enzymes evolved from existing bacterial components.Knox proposes that the penicillinase enzyme is the result of the
duplication of the D-Ala-D-Ala ligase protein that penicillin binds to.This explains the substrate specificity of the penicillinase enzyme.X-ray crystallography clearly demonstrates the striking "homologous
three-dimensional structures that extend well beyond the beta-lactam binding
site" - as well as similar catalytic activities.Knox goes on to say, "From these results, it seems likely that the
beta-lactamase evolved from an enzyme with DD-peptidase activity." 8

Of course, a hypothesis that "seems likely" is
very much different from one that is actually testable in the lab.
A lot of things might seem likely, but this is what makes something
"hypothetical." All hypothesis seem likely on
paper, but until they are tested, they remain hypothetical as far as the
scientific method is concerned.Knox showed some
seemingly reasonable hypothetical evolutionary pathways that could give rise to
the penicillinase enzyme, but none of these evolutionary pathways or portions
thereof was ever demonstrated by direct experimentation.If put to the test, they just might run into a few snags along the way. .
.

For example, it seems that not just one, but
several key changes are needed for "DD-peptidases" to achieve "beta-lactam
hydrolysis." It seems like a key substitution at "position
69" and a hydrogen bond from B3 to Asn 170 in the W-loop are fairly important
for lactamase activity. Knox's own evolutionary path for the simpler
"Class C" beta-lactamases from a DD-peptidase requires an additional mutation in
the YXN sequence. The more complicated "Class A" beta-lactamases require a
mutation in the SXN sequence plus the addition of a general base on the W-loop.
Knox describes some other "important differences which exist between the two
molecules [DD-peptidase and beta-lactamase] [that] must be accommodated by the
new binding site." These include the following: "Bonds about the nitrogen
atom of the b-lactam ring are nonplanar; the C6 b-acylamido group of the
b-lactam ring is quite apart in direction from the corresponding peptide group
of the DD-peptide; and the beta-lactam lacks a methyl group corresponding to the
penultimate D-methyl group of the DD-peptide." 8

Some might argue that DD-peptidases have a very
small amount of beta-lactamase activity to begin with. However, this is
not the point. The point is that it is not clear that each and every
mutation needed to achieve selectively advantageous beta-lactamase activity is
selectively advantageous. In other words, there might be some neutral gaps
in function that need to be crossed. Of course, potential neutral
mutations would not be selectively advantageous and therefore would take much
more time to randomly occur - especially if the neutral gap were more than two
or three mutations wide. Consider the follow amino acid sequences
structures for a DD-peptidase and a beta-lactamase: